PROJECT SUMMARY Staphylococcus aureus (S. aureus) is the leading cause of bacterial endocarditis. Methicillin-resistant S. aureus hospitalizations in the United States have more than doubled rendering traditional antibiotic treatments ineffective. Thus, understanding the factors modulating immune cell function at the host-pathogen interface is critical in identifying new therapeutic options. Several risk factors for S. aureus infection, including intravenous drug use, liver disease, or long-term intravenous nutrition, results in significantly increased levels of Mn in the tissues of patients. We have previously identified that elevated dietary manganese (Mn) promotes heightened S. aureus burdens in the heart following systemic infection coinciding with increased mortality of the host. Mn is a nutrient metal necessary for S. aureus growth, which is primarily acquired from the host. The host attempts to restrict nutrient metals from pathogens through a process termed `nutritional immunity'. Calprotectin (CP) is critical in maintaining nutritional immunity, since it is the only identified Mn-sequestering immunoprotein in vertebrates. Previously, we demonstrated that a deficiency in CP decreases the burdens of S. aureus in the heart suggesting that CP may play alternative roles in the heart independent of metal sequestration. Combined, this provides an ideal model to understand how dietary Mn and CP influence immune cell function during S. aureus infection. Preliminary data from our lab demonstrates that increasing the extracellular concentration of Mn or a CP deficiency alters neutrophil and macrophage function in response to S. aureus in vitro. Consistent with what was seen in the heart, increased concentrations of Mn prevents neutrophil killing of S. aureus in vitro. In contrast to the heart, CP-deficient neutrophils and macrophages have impaired S. aureus killing, which suggests that the heart provides a unique niche that is not accurately reproduced in cell culture conditions. Therefore, understanding the molecular mechanisms of Mn and CP on immune cell function is critical in understanding how dietary Mn and CP alters immune cell function in the heart during a systemic S. aureus infection. The central hypothesis of this proposal is that the mortality caused by elevated burdens of S. aureus in the heart is due to the altered immuno-homeostasis of elevated Mn and CP, which provides a niche for S. aureus colonization of the heart and enhanced bacterial virulence. This proposal will address this hypothesis by integrating in vitro and in vivo studies through a series of integrated aims. Aim 1 will determine how immune cell function is altered in mice on an elevated Mn diet. Aim 2 will elucidate the mechanism by which Mn promotes S. aureus colonization of the heart. Aim 3 will identify whether metal binding underlies CP enhancement of S. aureus infection of the heart. These studies are significant because they identify how minor alterations in dietary metals can alter the immune response. In addition, they will define how CP binding of Mn contributes to this process. Long-term, these studies could provide novel therapeutic options by modulating the immune response to pathogens through the alteration of dietary metals.